The present invention is concerned with assays (kits and analytical methods) for detecting or monitoring TSH Receptor Autoantibodies.
The hyperthyroidism which is associated with Graves' disease is known to be due to autoantibodies to the thyroidal receptor for thyroid stimulating hormone (TSH). The autoantibodies bind to the receptor and mimic the actions of the natural ligand (TSH), thereby causing the gland to produce high levels of thyroid hormones (as described in Endocrine Reviews 1988, Vol 9, No. 1, pages 106 to 117).
The detection or monitoring of TSH receptor autoantibodies (TRAb) is important in the diagnosis and management of Graves' disease and currently two types of assay are used, namely:                (a) competitive binding assays which measure the ability of TRAb to inhibit the binding of 125I-labelled TSH to preparations of TSH receptor; and        (b) bioassays which measure the ability of TRAb to stimulate thyroid cells (or other cells transfected with the TSH receptor gene) in culture.        
Currently, competitive binding assays (type (a) above) are more widely used, because bioassays of the type mentioned in (b) above are expensive, time-consuming, require highly skilled staff and are unsuitable for wide routine use. In current competitive binding assays, test serum samples (50 μl) are generally incubated with detergent solubilized porcine TSH receptor (50 μl). TRAb present in the test sera bind to the TSH receptor during this incubation. 125I-labelled TSH is then added and the incubation continued. During this second incubation, the labelled TSH binds to TSH receptors not already occupied by TRAb. Finally, 125I-labelled TSH bound to the receptor is separated from free labelled TSH by addition of polyethylene glycol (PEG), which precipitates the receptor bound TSH but not the free TSH. The radioactivity in the precipitates (separated by centrifugation) is then counted. In the assay, TRAb in test samples inhibits the binding of labelled TSH to the TSH receptor and this results in a lowering of the radioactivity in the precipitates. Assay results can be expressed as an index of inhibition of labelled TSH binding or by use of a set of assay calibrators.
The main limitations of this conventional assay are as follows:                (a) The assay measures competition between labelled TSH and the TSH receptor and may not detect TRAb which bind well to the receptor but in such a way as not to inhibit TSH binding strongly.        (b) The assay uses polyethylene glycol to separate receptor bound and free labelled TSH. This results in co-precipitation of all the serum immunoglobulins and the formation of a relatively large pellet. Although the pellet can be counted for radioactivity, it is not a suitable preparation to detect TSH (or other proteins or peptides) labelled with non-radioactive substances such as enzymes or chemiluminescent materials. This is because the serum components in the pellet interfere with such processes as light emission. In addition, the use of PEG precipitation necessitates the use of centrifugation and this is a time-consuming and cumbersome procedure unsuitable for automation.        
Published patent specification EP0719858A describes an assay method for TSH receptor autoantibodies, where TSH receptor is bound to a solid phase either directly or via anti-TSH receptor antibody. A method for determination of TSH receptor autoantibodies present in patient serum is also described in published patent specification WO95/06258.